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Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins
Author(s) -
Umetsu Mitsuo,
Tsumoto Kouhei,
Ashish Kumar,
Nitta Shigeki,
Tanaka Yoshikazu,
Adschiri Tadafumi,
Kumagai Izumi
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01441-8
Subject(s) - protein secondary structure , inclusion bodies , circular dichroism , pyrococcus horikoshii , native state , chemistry , denaturation (fissile materials) , guanidine , protein folding , biochemistry , protein tertiary structure , protein structure , recombinant dna , escherichia coli , protein aggregation , alpha helix , crystallography , biophysics , biology , enzyme , nuclear chemistry , gene
Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid‐state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The β‐strand‐rich single chain Fv fragment (scFv) and α‐helix‐rich interleukin (IL)‐4 lost part of the native‐like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is α‐helix rich, are predominated by α‐helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL‐4, whereas the solubilized hyperthermophilic proteins have retained the α‐helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native‐like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native‐like structure in inclusion bodies and suggests that non‐native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.